Fluorescent dyes for use in glucose sensing

ABSTRACT

A novel class of compounds that includes HPTS-Cys-MA, and methods of making them are disclosed herein. The class of compounds including HPTS-Cys-MA are useful as fluorescent dyes for analyte detection.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/833,081 filed Jul. 25, 2006 which is hereby incorporated by referencein its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Novel fluorescent dyes are disclosed for use in analyte detection.

2. Description of the Related Art

Fluorescent dyes, including 8-hydroxypyrene-1,3,6-trislfonic acid (HPTS)and its derivatives, are known and have been used in analyte detection.See e.g., U.S. Pat. Nos. 6,653,141, 6,627,177, 5,512,246, 5,137,833,6,800,451, 6,794,195, 6,804,544, 6,002,954, 6,319,540, 6,766,183,5,503,770, and 5,763,238; and co-pending U.S. patent application Ser.Nos. 10/456,895 and 11/296,898; each of which is incorporated herein inits entirety by reference thereto.

SUMMARY OF THE INVENTION

Fluorescent dyes having the below generic structure are disclosed inaccordance with embodiments of the present invention.

wherein:

-   R² is

-   R³ is —(CH₂)_(n)—A⁻M⁺,    -   wherein n is 1-4,    -   wherein A⁻ is an anionic group selected from the group        consisting of SO₃ ⁻, HPO₃ ⁻, CO₂ ⁻ and

and

-   -   wherein M⁺ is a cationic group selected from the group        consisting of H⁺, an alkali metal ion, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺,        Fr⁺, an onium ion and NR₄ ⁺, wherein R is selected from the        group consisting of alkyl, alkylaryl and aromatic groups);

-   R⁴ is

-   R⁵ is selected from the group consisting of

-   -   wherein n is equal to 1-10, n′ is equal to 2-4, and Y is        selected from the group consisting of NH and O;

-   R⁶ is selected from the group consisting of NHR⁷, OR⁷ and CO₂H; and

-   R⁷ is H or an ethylenically unsaturated group selected from the    group consisting of methacryloyl, acryloyl, styryl, acrylamido and    methacrylamido.

Fluorescent dyes having the below generic structure are disclosed inaccordance with preferred embodiments of the present invention.

wherein:

-   R³ is —(CH₂)_(n)—A⁻M⁺,    -   wherein n is 1-4,    -   wherein A⁻ is an anionic group selected from the group        consisting of SO₃ ⁻, HPO₃ ⁻, CO₂ ⁻ and

and

-   -   wherein M⁺ is a cationic group selected from the group        consisting of H⁺, an alkali metal ion, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺,        Fr⁺, an onium ion and NR₄ ⁺, wherein R is selected from the        group consisting of alkyl, alkylaryl and aromatic groups);

-   R⁸ is selected from the group consisting of

-   -   wherein n is equal to 1-10, n′ is equal to 2-4;

-   R⁹ is selected from the group consisting of NHR¹⁰, OR¹⁰ and CO₂H;    and

-   R¹⁰ is H or an ethylenically unsaturated group selected from the    group consisting of methacryloyl, acryloyl, styryl, acrylamido and    methacrylamido.

A fluorescent dye termed HPTS-Cys-MA (or HPTS-TriCys-MA) having thebelow structure is disclosed in accordance with preferred embodiments ofthe present invention.

A glucose sensor is disclosed in accordance with another embodiment ofthe present invention, comprising the dyes disclosed herein (e.g.,HPTS-Cys-MA) and a quencher comprising boronic acid, such as 3,3′-oBBV.

A first method of making the generic class of compounds to whichHPTS-Cys-MA belongs is disclosed in accordance with another embodimentof the present invention. The method comprises the following steps:

wherein:

-   R² is

-   R³ is —(CH₂)_(n)—A⁻M⁺,    -   wherein n is 1-4,    -   wherein A⁻ is an anionic group selected from the group        consisting of SO₃ ⁻, HPO₃ ⁻, CO₂ ⁻ and

and

-   -   wherein M⁺ is a cationic group selected from the group        consisting of H⁺, an alkali metal ion, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺,        Fr⁺, an onium ion and NR₄ ⁺, wherein R is selected from the        group consisting of alkyl, alkylaryl and aromatic groups);

-   R⁴ is

-   R⁵ is selected from the group consisting of

-   -   wherein n is equal to 1-10, n′ is equal to 2-4 and Y is selected        from the group consisting of NH and O;

-   R⁶ is selected from the group consisting of NHR⁷, OR⁷ and CO₂H;

-   R⁷ is H or an ethylenically unsaturated group selected from the    group consisting of methacryloyl, acryloyl, styryl, acrylamide and    methacrylamido; and

-   Z is an amino protecting group selected from the group consisting of    phthalimido, Boc and Fmoc).

A second method of making the generic class of compounds to whichHPTS-Cys-MA belongs is disclosed in accordance with another embodimentof the present invention. The method comprises the steps of:

wherein:

-   R³ is —(CH₂)_(n)—A⁻M⁺,    -   wherein n is 1-4,    -   wherein A⁻ is an anionic group selected from the group        consisting of SO₃ ⁻, HPO₃ ⁻, CO₂ ⁻ and

and

-   -   wherein M⁺ is a cationic group selected from the group        consisting of H⁺, an alkali metal ion, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺,        Fr⁺, an onium ion and NR₄ ⁺, wherein R is selected from the        group consisting of alkyl, alkylaryl and aromatic groups);

-   R⁸ is selected from the group consisting of

-   -   wherein n is equal to 1-10, n′ is equal to 2-4;

-   R⁹ is selected from the group consisting of NHR¹⁰, OR¹⁰ and CO₂H;    and

-   R¹⁰ is H or an ethylenically unsaturated group selected from the    group consisting of methacryloyl, acryloyl, styryl acrylamide and    methacrylamido.

A specific method for making HPTS-Cys-MA is disclosed in accordance withanother embodiment of the present invention. The method comprises thesteps of making HPTS-CysOH as follows:

and making HPTS-Cys-MA as follows:

Another specific method for making HPTS-Cys-MA is disclosed inaccordance with another embodiment of the present invention. The methodcomprises the steps of making:

a) TBCys as follows:

-   -   b) Phth acid as follows:

-   -   c) Phth MA as follows:

-   -   d) AminoCysMA as follows:

and

-   -   e) HPTS-Cys-MA as follows:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Stern-Volmer Quenching of HPTS-CysMA/3,3′-oBBV in Solution.Shows the relative emission change (Stern-Volmer curve) upon addition of3,3′-oBBV indicating the quenching of HPTS-CysMA with 3,3′-oBBV.

FIG. 2. Glucose Response of HPTS-CysMA/3,3′-oBBV in Solution. Shows thefluorescence emission measured after addition of glucose to HPTS-CysMAand 3,3′-oBBV.

FIG. 3. Fluorescence Spectra of HPTS-CysMA in Hydrogel. Showsfluorescence excitation and emission spectra of a polymer hydrogelcomprising HPTS-CysMA.

FIG. 4. Time Drive of HPTS-Cys-MA in hydrogel at Different pHs, Em=532nm. Shows time drive of HPTS-CysMA in hydrogel at different pH's.

FIG. 5. pH profile of HPTS-Cys-MA at two different excitations, Em=532nm. Shows pH profile of HPTS-CysMA at two different excitationwavelengths.

FIG. 6. Glucose Response of HPTS-CysMA/3,3′-oBBV in hydrogel. Shows theglucose response of HPTS-CysMA/3,3′-oBBV in a hydrogel.

FIG. 7 Fluorescence Spectra of HPTS-CysMA and HPTS-LysMA. Shows acomparison of the fluorescence spectra of HPTS-Cys-MA and HPTS-Lys-MA.(1×10⁻⁵ M); Ex Slit 8 nm, Em Slit 12 nm.

FIG. 8 Comparison of Stern-Volmer Quenching Using HPTS-LysMA andHPTS-CysMA with 3,3′-oBBV, [Dye]=1×10⁻⁵ M. Shows a comparison of3,3′-oBBV Stern-Volmer quenching study using HPTS-Cys-MA andHPTS-Lys-MA.

FIG. 9 Comparison of Glucose Modulation Using HPTS-LysMA and HPTS-CysMAwith 3,3′-oBBV; [Dye]=1×10⁻⁵ M; Q/D=150. Shows a comparison glucosemodulation using HPTS-Cys-MA and HPTS-Lys-MA with 3,3′-oBBV.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Fluorescent Dyes

The fluorescent dyes of the invention are derivatives of8-hydroxypyrene-1,3,6-trisulfonate (HPTS). The counterions can be H⁺ orany other cation. HPTS exhibits two excitation wavelengths at around 405nm and around 450 nm, which correspond to the absorption wavelengths ofthe acid and its conjugate base, respectively. The shift in excitationwavelength is due to the pH-dependent ionization of the hydroxyl groupon HPTS. As the pH increases, HPTS shows an increase in absorbance atabout 450 nm, and a decrease in absorbance below about 420 nm. ThepH-dependent shift in the absorption maximum enables dual-excitationratiometric detection in the physiological range. The dyes may be usedwith a quencher comprising boronic acid, such as 3,3′-oBBV.

A generic structure of dyes in accordance with preferred embodiments ofthe present invention is:

wherein:

-   R² is

-   R³ is —(CH₂)_(n)—A⁻M⁺,    -   wherein n is 1-4,    -   wherein A⁻ is an anionic group selected from the group        consisting of SO₃ ⁻, HPO₃ ⁻, CO₂ ⁻ and

and

-   -   wherein M⁺ is a cationic group selected from the group        consisting of H⁺, an alkali metal ion, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺,        Fr⁺, an onium ion and NR₄ ⁺, wherein R is selected from the        group consisting of alkyl, alkylaryl and aromatic groups);

-   R⁴ is

-   R⁵ is selected from the group consisting of

-   -   wherein n is equal to 1-10, n′ is equal to 2-4 and Y is selected        from the group consisting of NH and O;

-   R⁶ is selected from the group consisting of NHR⁷, OR⁷ and CO₂H; and

-   R⁷ is H or an ethylenically unsaturated group selected from the    group consisting of methacryloyl, acryloyl, styryl, acrylamide and    methacrylamido.

Another generic structure of dyes in accordance with preferredembodiments of the present invention is:

where:

-   R³ is —(CH₂)_(n)—A⁻M⁺,    -   wherein n is 1-4,    -   wherein A⁻ is an anionic group selected from the group        consisting of SO₃ ⁻, HPO₃ ⁻, CO₂ ⁻ and

and

-   -   wherein M⁺ is a cationic group selected from the group        consisting of H⁺, an alkali metal ion, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺,        Fr⁺, an onium ion and NR₄ ⁺, wherein R is selected from the        group consisting of alkyl, alkylaryl and aromatic groups);

-   R⁴ is

-   R⁸ is selected from the group consisting of

-   -   wherein n is equal to 1-10, n′ is equal to 2-4;

-   R⁹ is selected from the group consisting of NHR¹⁰, OR¹⁰ and CO₂H;    and

-   R¹⁰ is H or an ethylenically unsaturated group selected from the    group consisting of methacryloyl, acryloyl, styryl, acrylamido and    methacrylamido.

The structure of HPTS-Cys-MA is as follows:

As indicated in the generic structures above, substitutions other thanCys-MA on the HPTS core are consistent with aspects of the presentinvention, as long as the substitutions are negatively charged and havea polymerizable group. For example, either L or D stereoisomers ofcysteic acid may be used. In some embodiments, only one or two of thesulfonic acids may be substituted. Likewise, in variations to HPTS-CysMAshown above, other counterions besides NBu₄ ⁺ may be used, includingpositively charged metals, e.g., Na⁺. In other variations, the sulfonicacid groups may be replaced with e.g., phosphoric, carboxylic, etc.functional groups.

For comparison, the structure of HPTS-LysMA is pictured below asfollows:

First Method of Making the Generic Class of Compounds to whichHPTS-Cys-MA Belongs

A first method of making the generic class of compounds to whichHPTS-Cys-MA belongs is disclosed in accordance with another embodimentof the present invention. The method comprises the following steps:

wherein:

-   R² is

-   R³ is —(CH₂)_(n)—A⁻M⁺,    -   wherein n is 1-4,    -   wherein A⁻ is an anionic group selected from the group        consisting of SO₃ ⁻, HPO₃ ⁻, CO₂ ⁻ and

and

-   -   wherein M⁺ is a cationic group selected from the group        consisting of H⁺, an alkali metal ion, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺,        Fr⁺, an onium ion and NR₄ ⁺, wherein R is selected from the        group consisting of alkyl, alkylaryl and aromatic groups);

-   R⁴ is

-   R⁵ is selected from the group consisting of

-   -   wherein n is equal to 1-10, n′ is equal to 2-4 and Y is selected        from the group consisting of NH and O;

-   R⁶ is selected from the group consisting of NHR⁷, OR⁷ and CO₂H;

-   R⁷ is H or an ethylenically unsaturated group selected from the    group consisting of methacryloyl, acryloyl, styryl, acrylamide and    methacrylamido; and

-   Z is an amino protecting group selected from the group consisting of    phthalimido, Boc and Fmoc).    Second Method of Making the Generic Class of Compounds to which    HPTS-Cys-MA Belongs

A second method of making the generic class of compounds to whichHPTS-Cys-MA belongs is disclosed in accordance with another embodimentof the present invention. The method comprises the steps of:

wherein:

-   R³ is —(CH₂)_(n)—A⁻M⁺,    -   wherein n is 1-4,    -   wherein A⁻ is an anionic group selected from the group        consisting of SO₃ ⁻, HPO₃ ⁻, CO₂ ⁻ and

and

-   -   wherein M⁺ is a cationic group selected from the group        consisting of H⁺, an alkali metal ion, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺,        Fr⁺, an onium ion and NR₄ ⁺, wherein R is selected from the        group consisting of alkyl, alkylaryl and aromatic groups);

-   R⁸ is selected from the group consisting of

-   -   wherein n is equal to 1-10, n′ is equal to 2-4;

-   R⁹ is selected from the group consisting of NHR¹⁰, OR¹⁰ and CO₂H;    and

-   R¹⁰ is H or an ethylenically unsaturated group selected from the    group consisting of methacryloyl, acryloyl, styryl,acrylamide and    methacrylamido.    First Specific Method of Synthesizing HPTS-Cys-MA

Cysteic acid 1 (0.5 mmols, 94 mg-L stereoisomer was used in thissynthesis; however, D stereoisomer may also be used) was treated with anaqueous solution of tetrabutylammonium hydroxide (0.5 mmols, 4 mL of0.125 M solution) at room temperature (scheme 1). After stirring for 30min, the solution was lyophilized to give 2 and the residue wasdissolved in dichloromethane (2 mL). Triethylamine (0.6 mmols, 61 mg)was added followed by the dropwise addition of HPTS-Cl (0.1 mmols, 52mg) in dichloromethane (2 mL). The mixture stirred for 18 h at roomtemperature and was then concentrated in vacuo. The residue wasdissolved in hot isopropyl alcohol and loaded onto a Biotage SP125Msilica gel cartridge and eluted with NH₄OH:isopropyl alcohol (1:3) togive HPTS-CysOH as a yellow powder (0.024 mmols, 39 mg, 24%). ¹H NMR(D₂O, 500 MHz) δ 0.85 (t, J=7.4 Hz, 36H), 1.20 (s, J=7.4 Hz, 24H) 1.44(m, 24H), 2.94 (m, 24H), 3.24 (m, 6H), 4.31 (m, 3H), 8.19 (d, J=16.8 Hz,2H), 8.36 (d, J=9.0 Hz, 1H), 8.50 (d, J=9.3 Hz, 1H), 8.64 (d, J=9.3 Hz,1H), 8.71 (d, J=7.1 Hz, 1H), 8.90 (m, 4H), 9.19 (d, J=12.0 Hz, 2H).

To a solution of HPTS-CysOH (0.0183 mmols, 30 mg) in DMF (1 mL) wasadded N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)(0.055 mmols 10.5 mg), 1-hydroxybenzotriazole (HOBT) (0.055 mmols, 7.4mg), N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate (HBTU) (0.055 mmols, 21 mg), andN,N′-diisopropylethylamine (DIEA) (0.183 mmols, 24 mg). After thesolution was stirred at room temperature for 20 min,N-(3-aminopropyl)methacrylamide hydrochloride was added and the mixturewas stirred for 48 h. The reaction mixture was precipitated withacetone:ether (5:1, 10 mL) to give an oily residue. The residue wastriturated with acetone (10 mL) and sonicated for 30 min to giveHPTS-CysMA as a crude mixture (35 mg of orange powder).

Second Specific Method of Synthesizing HPTS-Cys-MA

Step 1: Synthesis of TBCys:

In a 500-mL beaker equipped with a magnetic stirring bar, (L)-Cysteicacid (129.24 mmols, 24.1913 g) was dissolved in H₂O (100 mL) and treatedwith tetrabutylammonium hydroxide (129.24 mmols, 129.24 mL, 128.59 g ofa 1.0 M aqueous solution). The mixture was stirred at room temp for 30min and then frozen using a low temperature (dry ice/2-propanol) bath.The solid was lyophilized over three days to give a glassy material thatwas re-dissolved in CH₂Cl₂ (300 mL). The solution was evaporated todryness in a 500-mL flask to give TBCys as a white foam. Yield: 53.3756g, 129 mmols, 100%.

Step 2: Synthesis of Phth acid:

In a 500-mL flask equipped with a magnetic stirring bar, TBCys (129mmols, 53.1936 g) was dissolved in CHCl₃ (129 mL), andN,N′-diisopropylethyl amine (65 mmols, 8.385 g, 11.3 mL) was addedfollowed by phthalic anhydride (129 mmols, 19.10748 g) and 4-angstrommolecular sieves (50 mL); the mixture was refluxed for 48 h. The mixturewas filtered through a 600-mL glass fritted funnel and the filtrate wasconcentrated in vacuo and washed with diethyl ether (2×100 mL) and driedunder reduced pressure to give Phth acid complexed withN,N′-diisopropylethyl amine. Yield: 70 g, 129 mmols, 100%. ¹H NMR(CDCl₃, 500 MHz) δ 0.95 (t, J=7.4 Hz, 12H), 1.30 (m, 8H), 1.38 (q, J=7.4Hz, 8H), 1.61 (m, 8 H), 2.96 (q, J=7.4 Hz, 1H), 3.23 (m, 8H), 3.55(sept, J=6.7, 1H), 3.62 (m, 1H), 3.86 (dd, J₁=5 Hz, J₂=9.4 Hz, 1H), 5.31(dd, J₁=5.4 Hz, J₂=1.2 Hz, 1H), 7.65 (q, J=3 Hz, 2H), 7.55 (q, J=3 Hz,2H); ¹³C NMR (CDCl₃, 125 MHz) δ 12.0, 13.8, 18.1, 19.8, 24.0, 41.7,50.4, 51.0, 53.1, 58.7, 123.0, 132.8, 133.52, 167.9, 170.7; HPLCsupelcosil LC-8-DB, 5 μm, 150 mm×4.6 mm, λ=254 nm, gradient elution (30to 70% MeOH) with MeOH/TBAP, R_(t)=5.9 min.

Step 3: Synthesis of Phth MA:

In a 250-mL round bottom flask equipped with a magnetic stirring bar,Phth acid (24.9 mmols, 13.46 g) was dissolved in CH₂Cl₂ (85 mL) at 0° C.and EDC (27 mmols, 5.157 g), HOBT (27 mmols, 3.645 g), and triethylamine(54 mmols, 5.4 g, 7.5 mL) were added sequentially; the mixture wasstirred under nitrogen for 20 min. N-(3-Aminopropyl)methacrylamidehydrochloride (27 mmols, 4.806 g) was added and the mixture was allowedto reach room temperature over 2 h and then was stirred for anadditional 14 h at room temp. The solution was treated with sat. NaHCO₃(100 mL) and extracted with CH₂Cl₂ (3×20 mL). The CH₂Cl₂ layer was driedover MgSO₄ and concentrated in vacuo to give an orange oil that wasloaded onto a Biotage KP-sil 40M cartridge. The material was purifiedvia gradient elution with 5 to 15% MeOH/CH₂Cl₂. The product was isolatedas a white foam, yield 72%. ¹H NMR (CDCl₃, 500 MHz) δ 0.99 (t, J=7.4 Hz,12H), 1.44 (sext, J=7.4 Hz, 8H), 1.66 (m, 10H), 1.89 (s, 3H), 3.28 (m,11H), 3.38 (sext, 2H), 3.96 (dd, J₁=9 Hz, J₂=5.9 Hz, 1H), 5.22 (t, J=1.4Hz, 1H), 5.44 (dd, J₁=1.9 Hz, J₂=9.0 Hz, 1H), 5.69 (s, 1H), 5.91 (t,J=6.0 Hz, 1H), 7.70 (m, 2H), 7.82 (m, 2H), 9.09 (t, j=5.8 Hz, 1H); ¹³CNMR (CDCl₃, 125 MHz) δ 13.8, 18.7, 19.8, 24.1, 29.1, 36.0, 36.4, 50.5,53.2, 58.9, 119.6, 123.4, 132.3, 134.0, 140.0, 167.9, 168.5, 169.3; HPLCsupelcosil LC-8-DB, 5 μm, 150 mm×4.6 mm, λ=254 nm, gradient elution (30to 70% MeOH) with MeOH/TBAP, R_(t)=6.6 min.

Step 4: Synthesis of AminoCysMA:

In a 100-mL round bottom flask equipped with a magnetic stirring bar,PhthMA (2.59 mmols, 1.7225 g) was dissolved in ethanol (20 mL) andhydrazine monohydrate (2.59 mmols, 0.130 g, 0.126 mL) was added and themixture was heated at 80° C. for two hours. The solution was cooled toroom temperature and CH₂Cl₂ (20 mL) was added. The precipitate that wasformed was filtered using a fritted funnel and washed with additionalCH₂Cl₂ (10 mL) and the filtrate was concentrated in vacuo. The residuewas dissolved in CH₂Cl₂ (5 mL) and loaded onto a Biotage KP-NH 40Mcartridge and eluted with 1% to 15% MeOH/CH₂Cl₂. The fractions werecollected and concentrated in vacuo to give aminoCysMA as a colorlessoil. Yield: 60%. ¹H NMR (CD₃OD, 500 MHz) δ 1.03 (t, J=7.4 Hz, 12H), 1.42(sext, J=7.4 Hz, 8H), 1.66 (m, 8H), 1.95 (s, 3H), 2.87 (q, J₁=9.4 Hz,J₂=4.4 Hz, 1H), 3.23 (m, 10H), 3.81 (dd, J₁=3.3 Hz, J₂=6.0 Hz, 1H), 5.37(s, 1H), 5.73 (s, 1H); HPLC supelcosil LC-8-DB, 5 μm, 150 mm×4.6 mm,λ=254 nm, gradient elution (30 to 70% MeOH) with MeOH/TBAP, R_(t)=3.5min.

Step 5: Synthesis of HPTS-Cys-MA:

In a 50-mL round bottom flask, AminoCysMA (1.09 mmols, 0.5827 g) wasdissolved in CH₂Cl₂ (10 mL) and HPTS-Cl (0.29 mmols, 0.163 g) was added.Triethylamine (1.1 mmols, 0.153 mL) was added and the mixture wasstirred at room temperature for 16 h. The red solution was treated with1 M NaOH (10 mL), stirred for 30 min., and the two layers were separatedin a separatory funnel. The orange-aqueous layer was passed through acolumn of Dowex 50W resin (in the H⁺ form) to give a yellow/greensolution with a pH=4 as determined by pH paper. The aqueous solution wasthen passed through a column of Dowex 50W (in the Na⁺ form) to obtainthe crude sodium salt of HPTS-CysMA. The solution was adsorbed ontopolystyrene-divinylbenzene resin (250 g) and washed with H₂O (5×500 mL).The washings were kept and the adsorbed material was removed from theresin with MeOH (1 L). The MeOH/water extract was evaporated to drynessafter multiple co-evaporations with fresh MeOH (4×500 mL) and theresidue was re-dissolved in MeOH (0.5 mL). Acetone (15 mL) was added andthe precipitate was collected by centrifugation and dried under a streamof argon to give HPTS-Cys-MA as an orange/yellow solid. Additionalproduct was isolated from the washings using the same MeOH-extractionprocedure. Yield: 40%. ¹H-NMR (500 MHz, D2O, ppm): 1.18-1.09 (m, 6H),1.76-1.53 (m, 9H), 2.73-2.23 (m, 12H); HPLC supelcosil LC-8-DB, 5 μm,150 mm×4.6 mm, FLD detector, gradient elution (30 to 70% MeOH) withMeOH/TBAP, R_(t)=11.6 min; MALDI-TOF for C₄₆H₅₈N₉Na₃O₂₂S₆ [MH]⁺: 1350,[MH-Na+H]⁺:1328, [MH-2Na+2H]⁺: 1306, [MH-3Na+3H]⁺: 1284 (major).

Quenchers

As used herein, the term “quencher” refers to a compound that reducesthe emission of a fluorescent dye, e.g., HPTS-Cys-MA, when in itspresence.

In some embodiments, a quencher moiety provides glucose recognition.Such moieties preferably comprise an aromatic boronic acid. Morespecifically, the boronic acid is covalently bonded to a conjugatednitrogen-containing heterocyclic aromatic bis-onium structure (e.g., aviologen) in which the boronic acid reacts reversibly or irreversiblywith glucose in aqueous, organic or combination media to form boronateesters. The extent of the reaction is related to glucose concentrationin the medium.

Bis-onium salts are prepared from conjugated heterocyclic aromaticdinitrogen compounds. The conjugated heterocyclic aromatic dinitrogenare, e.g., dipyridyls, dipyridyl ethylenes, dipyridyl phenylenes,phenanthrolines, and diazafluorenes. It is understood that all isomersof said conjugated heterocyclic aromatic dinitrogen compounds in whichboth nitrogens can be substituted are useful in this invention. Boronicacid-substituted viologens and boronic acid-substituted polyviologensare described in detail in co-pending U.S. application Ser. No.11/671,880; incorporated herein in its entirety by reference thereto.

In other embodiments, pyridinium boronic acid quenchers are used incombination with the dyes of the present invention. Pyridinium boronicacid quenchers are described in detail in U.S. Provisional ApplicationNo. 60/915,372; incorporated herein in its entirety by referencethereto.

In one preferred embodiment, 3,3′-oBBV may be used as a quencher moiety.The structure of 3,3′-oBBV is:

Functional Analysis of Dyes

HPTS-Cys-MA was tested in solution using the fluorimeter to determinehow it compares with HPTS-LysMA. Stern-Volmer and glucose responsestudies were carried out back to back under identical conditions toensure direct comparison.

Solution Studies

To a solution of HPTS-CysMA (1×10⁻⁵ M in pH 7.4 PBS) was addedincreasing amounts of 3,3′-oBBV (30 mM in MeOH) and the fluorescenceemission measured after each addition. FIG. 1 gives the relativeemission change (Stern-Volmer curve) upon addition of 3,3′-oBBVindicating the quenching of HPTS-CysMA with 3,3′-oBBV. The fluorimetersettings were as follows: 1% attenuation, ex slit 8 nm, em slit 12 nm,486 nm ex λ, 537 nm em λ.

HPTS-CysMA (1×10⁻⁵ M) and 3,3′-oBBV (3×10⁻³ M) were titrated with astock solution of glucose (31250 mg/dL) in pH 7.4 PBS and thefluorescence emission measured after each addition of glucose Therelative change upon addition of glucose is given in FIG. 2.

Polymer Studies

HPTS-CysMA (1 mg), N,N′-dimethylacrylamide (400 mg),N,N′-methylenebisacrylamide (8 mg), HCl (10 μL of 1 M solution), andVA-044 (1 mg) were dissolved in water and diluted to 1 mL in avolumetric flask. The solution was freeze-pump-thawed (3×), injectedinto a mold containing a 0.005″ polyimide spacer and polymerized at 55°C. for 16 h. The resultant film was placed in pH 7.4 phosphate bufferand its fluorescence excitation and emission spectra obtained (FIG. 3).

The film was tested in a flow cell configuration at various pHs(initially at pH 5.1; changed to pH 5.75, 6.26, 6.5, 6.9, 7.4, 8.02, andthen back to pH 7.4) at two different excitation wavelengths (418 nm and486 nm) and was monitored at one emission wavelength (532 nm) over time(FIG. 4). The fluorimeter settings were as follows: ex slit 5 nm, emslit 3.5 nm, 515 nm cutoff filter, 418 nm ex λ, 486 nm ex λ, 532 nm emλ. The pH profile is summarized in FIG. 5.

HPTS-CysMA (2 mg), 3,3′-oBBV (15 mg), N,N′-dimethylacrylamide (400 mg),N,N′-methylenebisacrylamide (8 mg), HCl (10 μL of 1 M solution), andVA-044 (1 mg) were dissolved in water and diluted to 1 mL in avolumetric flask. The solution was freeze-pump-thawed (3×), injectedinto a mold containing a 0.005″ polyimide spacer and polymerized at 55°C. for 16 h. The resultant film was placed in pH 7.4 phosphate bufferand was tested in a flow cell configuration with increasing amounts ofglucose (0, 50, 100, 200, 400 mg/dL). The relative fluorescence changeupon addition of glucose is given in FIG. 6. The fluorimeter settingswere as follows: ex slit 8 nm, em slit 3.5 nm, 515 nm cutoff filter, 486nm ex λ, 532 nm em λ.

Comparison Studies

A comparison of the fluorescence spectra of the CysMA and LysMA dyes insolution is shown in FIG. 7. The CysMA dye is blue-shifted relative tothe LysMA dye.

A comparison of the Stem-Volmer quenching study for CysMA and LysMA insolution is given in FIG. 8 and the glucose response is given in FIG. 9.

HPTS-CysMA is quenched more effectively with 3,3′-oBBV than isHPTS-LysMA. HPTS-CysMA forms a stronger complex because of the sulfonicacids than does HPTS-LysMA, which has carboxylic acids; the tightercomplex leads to a greater glucose response. For this reason, dyessubstituted with sulfonate groups are preferred.

Glucose Sensors

In one preferred embodiment, a device is disclosed for determining bloodglucose concentration. The device comprises a sensor comprising anoptical fiber sized for deployment intravascularly. The sensor furthercomprises a water-insoluble polymer matrix, wherein the polymer matrixis permeable to glucose; a fluorescent dye, as disclosed herein,associated with the polymer matrix; a quencher as disclosed herein,adapted to reversibly bind an amount of glucose related to the bloodglucose concentration, wherein the quencher is also associated with thepolymer matrix and operably coupled to the fluorescent dye, and whereinthe quencher is configured to modulate the light emitted by thefluorescent dye related to the amount of bound glucose; at least oneexcitation light source; and an emission light detector.

A method is also disclosed for determining blood glucose concentration.The method comprises the steps of: providing the device described above;inserting the sensor into a blood vessel; irradiating the sensor at anexcitation wavelength; detecting a fluorescence emission of the sensorat an emission wavelength; and determining the blood glucoseconcentration.

In some embodiments, for use in vitro not involving a moving stream, thesensing components are used as individual (discrete) components. The dyeand quencher are mixed together in liquid solution, analyte is added,the change in fluorescence intensity is measured, and the components arediscarded. Polymeric matrices that can be used to trap the sensingcomponents to prevent leaching need not be present. Optionally, thesensing components are immobilized which allows their use to measureanalytes in a moving stream.

For in vivo applications, the sensor is used in a moving stream ofphysiological fluid, preferably blood, which contains one or morepolyhydroxyl organic compounds or is implanted in tissue such as musclewhich contains said compounds. Therefore, it is preferred that none ofthe sensing moieties escape from the sensor assembly. Thus, for use invivo, the sensing components are preferably part of an organic polymersensing assembly. Soluble dyes and quenchers can be confined by asemi-permeable membrane that allows passage of the analyte but blockspassage of the sensing moieties. This can be realized by using solublesensing moieties that are substantially larger than the analytemolecules (molecular weight of at least twice that of the analyte orgreater than 1000 preferably greater than 5000); and employing aselectively semipermeable membrane such as a dialysis or anultrafiltration membrane with a specific molecular weight cutoff betweenthe two so that the sensing moieties are quantitatively retained.

Preferably the sensing moieties are immobilized in an insoluble polymermatrix, which is freely permeable to glucose. The polymer matrix iscomprised of organic, inorganic or combinations of polymers thereof. Thematrix may be composed of biocompatible materials. Alternatively, thematrix is coated with a second biocompatible polymer, and/or asemipermeable membrane, that is permeable to the analytes of interest.

The function of the polymer matrix is to hold together and immobilizethe fluorescent dye and quencher moieties while at the same timeallowing contact with the analyte, and binding of the analyte to theboronic acid. To achieve this effect, the matrix must be insoluble inthe medium, and in close association with it by establishing a highsurface area interface between matrix and analyte solution. For example,an ultra-thin film or microporous support matrix is used. Alternatively,the matrix is swellable in the analyte solution, e.g. a hydrogel matrixis used for aqueous systems. In some instances, the sensing polymers arebonded to a surface such as the surface of a light conduit, orimpregnated in a microporous membrane. In all cases, the matrix must notinterfere with transport of the analyte to the binding sites so thatequilibrium can be established between the two phases. Techniques forpreparing ultra-thin films, microporous polymers, microporous sol-gels,and hydrogels are established in the art. All useful matrices aredefined as being analyte permeable.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications, and publications, referredto above, are hereby incorporated by reference.

1. The compound:

wherein: R² is

R³ is —(CH₂)_(n)—A⁻M⁺, wherein n is 1-4, wherein A⁻ is an anionic groupselected from the group consisting of SO₃ ⁻, HPO₃ ⁻, CO₂ ⁻ and

and wherein M⁺ is a cationic group selected from the group consisting ofH⁺, an alkali metal ion, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, Fr⁺, an onium ion andNR₄ ⁺, wherein R is selected from the group consisting of alkyl,alkylaryl and aromatic groups); R⁴ is

R⁵ is selected from the group consisting of

wherein n is equal to 1-10, n′ is equal to 2-4 and Y is selected fromthe group consisting of NH and O; R⁶ is selected from the groupconsisting of NHR⁷, OR⁷ and CO₂H; and R⁷ is H or an ethylenicallyunsaturated group selected from the group consisting of methacryloyl,acryloyl, styryl, acrylamido and methacrylamido.
 2. The compound:

wherein: R³ is —(CH₂)_(n)—A⁻M⁺, wherein n is 1-4, wherein A⁻ is ananionic group selected from the group consisting of SO₃ ⁻, HPO₃ ⁻, CO₂ ⁻and

and wherein M⁺ is a cationic group selected from the group consisting ofH⁺, an alkali metal ion, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, Fr⁺, an onium ion andNR₄ ⁺, wherein R is selected from the group consisting of alkyl,alkylaryl and aromatic groups); R⁴ is

R⁸ is selected from the group consisting of

wherein n is equal to 1-10, n′ is equal to 2-4; R⁹ is selected from thegroup consisting of NHR¹⁰, OR¹⁰ and CO₂H; and R¹⁰ is H or anethylenically unsaturated group selected from the group consisting ofmethacryloyl, acryloyl, styryl, acrylamido and methacrylamido.
 3. Thecompound:


4. The compound of claim 3, wherein NBu₄ is any other counterion.
 5. Thecompound of claim 3, comprising L, D, or L and D stereoisomers ofcysteine.
 6. A glucose sensor comprising the compound of claim
 1. 7. Theglucose sensor of claim 6, further comprising a quencher moiety.
 8. Theglucose sensor of claim 7, wherein said quencher moiety comprisesboronic acid.
 9. The glucose sensor of claim 8, wherein said boronicacid quencher is 3,3′-oBBV.
 10. A method of making the compound of claim1, comprising the steps of:

wherein: R² is

R³ is —(CH₂)_(n)—A⁻M⁺, wherein n is 1-4, wherein A⁻ is an anionic groupselected from the group consisting of SO₃ ⁻, HPO₃ ⁻, CO₂ ⁻ and

and wherein M⁺ is a cationic group selected from the group consisting ofH⁺, an alkali metal ion, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, Fr⁺, an onium ion andNR₄ ⁺, wherein R is selected from the group consisting of alkyl,alkylaryl and aromatic groups); R⁴ is

R⁵ is selected from the group consisting of

wherein n is equal to 1-10, n′ is equal to 2-4 and Y is selected fromthe group consisting of NH and O; R⁶ is selected from the groupconsisting of NHR⁷, OR⁷ and CO₂H; R⁷ is H or an ethylenicallyunsaturated group selected from the group consisting of methacryloyl,acryloyl, styryl, acrylamido and methacrylamido; and Z is an aminoprotecting group selected from the group consisting of Phthalimido, Bocand Fmoc.
 11. A method of making the compound of claim 2, comprising thesteps of:

wherein: R³ is —(CH₂)_(n)—A⁻M⁺, wherein n is 1-4, wherein A⁻ is ananionic group selected from the group consisting of SO₃ ⁻, HPO₃ ⁻, CO₂ ⁻and

and wherein M⁺ is a cationic group selected from the group consisting ofH⁺, an alkali metal ion, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, Fr⁺, an onium ion andNR₄ ⁺, wherein R is selected from the group consisting of alkyl,alkylaryl and aromatic groups); R⁸ is selected from the group consistingof

wherein n is equal to 1-10, n′ is equal to 2-4; R⁹ is selected from thegroup consisting of NHR¹⁰, OR¹⁰ and CO₂H; and R¹⁰ is H or anethylenically unsaturated group selected from the group consisting ofmethacryloyl, acryloyl, styryl, acrylamido and methacrylamido.
 12. Amethod of making the compound of claim 3, comprising the steps of makingHPTS-CysOH as follows; and

making HPTS-Cys-MA as follows:


13. A method of making the compound of claim 3, comprising the steps of:making TBCys as follows:

making Phth acid as follows:

making Phth MA as follows:

making AminoCysMA as follows:

and making HPTS-Cys-MA as follows: